Methods and pharmaceutical compositions for the treatment of bacterial superinfections post-influenza

ABSTRACT

The present invention relates to methods and pharmaceutical compositions for the treatment of bacterial superinfections post-influenza. In particular, the present invention relates to a method of treating a bacterial superinfection post-influenza in a subject in need thereof comprising administering the subject with a therapeutically effective amount of a flagellin polypeptide optionally in combination with at least one antibiotic.

FIELD OF THE INVENTION

The present invention relates to methods and pharmaceutical compositionsfor the treatment of bacterial superinfections post-influenza.

BACKGROUND OF THE INVENTION

Influenza A virus (IAV) infection is one of the most important causes ofrespiratory tract diseases and is responsible for widespread morbidityand mortality. During the first several days after infection, the hostdevelops a complex and effective innate immune response that allows tocontain IAV replication pending the development of adaptive immuneresponses. However, at later time points, increased susceptibility tobacterial superinfection can occur leading to mortality during IAVepidemics and pandemics. For instance bacterial pneumonias accounted forthe majority of deaths (˜50 million deaths worldwide) in the 1918pandemic (Spanish flu). Among the predominant bacteria species causingbacterial superinfection post-IAV are Streptococcus pneumoniae (thepneumococcus), Haemophilus influenzae and Staphylococcus aureus. Thus,there is a need for treatment of bacterial superinfectionspost-influenza. Although there are evidences of specific features ofindividual types of bacteria, the mechanisms leading to enhancesusceptibility to secondary bacterial infection seem to be broad-basedand include alterations of mechanical and immunological defences.Indeed, alteration of the physical barriers to bacterial adhesion andinvasion including alteration of the mucosa as well as the exposition ofnew attachment sites for the bacteria have been described. In parallel,impairment of the host innate (rather than adaptive) response is acardinal feature of bacterial-associated pneumonia post-influenzachallenge. There are now strong evidences that TLRS signaling inducesprotective mechanisms against bacterial infections. For instance, it wasalso showed that mucosal administration of flagellin into mice couldprotect against Streptococcus pneumoniae lung infection. Theanti-infectious properties of flagellin were mainly observed when theTLRS ligand was co-administrated with the pathogen or 2 to 24 h beforethe bacterial challenge (Munoz N, Van Maele L, Marques J M, Rial A,Sirard J C, Chabalgoity J A. Mucosal administration of flagellinprotects mice from Streptococcus pneumoniae lung infection. Infect Immun2010; 78:4226-33).

SUMMARY OF THE INVENTION

The present invention relates to methods and pharmaceutical compositionsfor the treatment of bacterial superinfections post-influenza. Inparticular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION

The inventors investigated the effectiveness of a combination therapyconsisting of flagellin+antibiotic on IAV-infected animals. For thispurpose, animals infected with IAV for 7 days were infected with S.pneumoniae and treated with AMX and flagellin. They show that thecombination therapy was highly effective to increase the therapeuticindex of AMX both in lungs and spleen.

The present invention relates to a method of treating a bacterialsuperinfection post-influenza in a subject in need thereof comprisingadministering the subject with a therapeutically effective amount of aflagellin polypeptide optionally in combination with at least oneantibiotic.

The subject can be human or any other animal (e.g., birds and mammals)susceptible to influenza infection (e.g. domestic animals such as catsand dogs; livestock and farm animals such as horses, cows, pigs,chickens, etc.). Typically said subject is a mammal including anon-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep,cat, dog, rat, and mouse) and a primate (e.g., a monkey, chimpanzee, anda human). In some embodiments, the subject is a human.

According to the invention the subject suffers or has suffered from aninfluenza infection. As used herein, the term “influenza infection” hasits general meaning in the art and refers to the disease caused by aninfection with an influenza virus. In some embodiments of the invention,influenza infection is associated with Influenza virus A or B. In someembodiments of the invention, influenza infection is associated withInfluenza virus A. In some specific embodiments of the invention,influenza infection is cause by influenza virus A that is H1N1, H2N2,H3N2 or H5N1.

As used herein, the term “bacterial superinfection post-influenza” hasits general meaning in the art and refers to a bacterial infection (e.g.bacterial pneumonia) which occurs in a subject who suffers or hassuffered from an influenza infection. Typically, the bacterialsuperinfection occurs within 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days after influenzainfection. The method of the present invention is particularly suitablefor the treatment of a bacterial superinfection post-influenza such as,but not limited to infections of the lower respiratory tract (e.g.,pneumonia), middle ear infections (e.g., otitis media) and bacterialsinusitis. The bacterial superinfection may be caused by numerousbacterial pathogens. For example, they may be mediated by at least oneorganism selected from the group consisting of: Streptococcuspneumoniae; Staphylococcus aureus; Haemophilus influenza, Myoplasmaspecies and Moraxella catarrhalis.

As used herein, the term “treatment” or “treat” refer to bothprophylactic or preventive treatment as well as curative or diseasemodifying treatment, including treatment of patient at risk ofcontracting the disease or suspected to have contracted the disease aswell as patients who are ill or have been diagnosed as suffering from adisease or medical condition, and includes suppression of clinicalrelapse. The treatment may be administered to a subject having a medicaldisorder or who ultimately may acquire the disorder, in order toprevent, cure, delay the onset of, reduce the severity of, or ameliorateone or more symptoms of a disorder or recurring disorder, or in order toprolong the survival of a subject beyond that expected in the absence ofsuch treatment. By “therapeutic regimen” is meant the pattern oftreatment of an illness, e.g., the pattern of dosing used duringtherapy. A therapeutic regimen may include an induction regimen and amaintenance regimen. The phrase “induction regimen” or “inductionperiod” refers to a therapeutic regimen (or the portion of a therapeuticregimen) that is used for the initial treatment of a disease. Thegeneral goal of an induction regimen is to provide a high level of drugto a patient during the initial period of a treatment regimen. Aninduction regimen may employ (in part or in whole) a “loading regimen”,which may include administering a greater dose of the drug than aphysician would employ during a maintenance regimen, administering adrug more frequently than a physician would administer the drug during amaintenance regimen, or both. The phrase “maintenance regimen” or“maintenance period” refers to a therapeutic regimen (or the portion ofa therapeutic regimen) that is used for the maintenance of a patientduring treatment of an illness, e.g., to keep the patient in remissionfor long periods of time (months or years). A maintenance regimen mayemploy continuous therapy (e.g., administering a drug at a regularintervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy(e.g., interrupted treatment, intermittent treatment, treatment atrelapse, or treatment upon achievement of a particular predeterminedcriteria [e.g., disease manifestation, etc.]).

The method of the present invention is particularly suitable forsubjects who are identified as at high risk for developing a bacterialsuperinfection post-influenza, including subjects who are at least 50years old, subjects who reside in chronic care facilities, subjects whohave chronic disorders of the pulmonary or cardiovascular system,subjects who required regular medical follow-up or hospitalizationduring the preceding year because of chronic metabolic diseases(including diabetes mellitus), renal dysfunction, hemoglobinopathies, orimmunosuppression (including immunosuppression caused by medications orby human immunodeficiency [HIV] virus); children less than 14 years ofage, patients between 6 months and 18 years of age who are receivinglong-term aspirin therapy, and women who will be in the second or thirdtrimester of pregnancy during the influenza season. More specifically,it is contemplated that the method of the invention is suitable for thetreatment of bacterial superinfection post-influenza in subjects olderthan 1 year old and less than 14 years old (i.e., children); subjectsbetween the ages of 50 and 65, and adults who are older than 65 years ofage.

As used herein, the term “flagellin” has its general meaning in the artand refers to the flagellin contained in a variety of Gram-positive orGram-negative bacterial species. Non-limiting sources of flagellinsinclude but are not limited to Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella entericaserovar Typhimurium, Serratia, e.g., Serratia marcescans, and Shigella,as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonassuch as P. aeruginosa, and Streptomyces. These examples are illustrativerather than limiting. The amino acid sequences and nucleotide sequencesof flagellins are publically available in the NCBI Genbank, see forexample Accession Nos. AAL20871, NP_310689, BAB58984, AAO85383,AAA27090, NP_461698, AAK58560, YP_001217666, YP_002151351, YP_001250079,AAA99807, CAL35450, AAN74969, and BAC44986. The flagellin sequences fromthese and other species are intended to be encompassed by the termflagellin as used herein. Therefore, the sequence differences betweenspecies are included within the meaning of the term.

The term “flagellin polypeptide” is intended to a flagellin or afragment thereof that retains the ability to bind and activate TLRS. Asused herein the term “toll-like receptor 5” or “TLRS” has its generalmeaning in the art and is intended to mean a toll-like receptor 5 of anyspecies, but preferably a human toll-like receptor 5. Upon activation, aTLRS induces a cellular response by transducing an intracellular signalthat is propagated through a series of signaling molecules from the cellsurface to the nucleus. Typically, the intracellular domain of TLRSrecruits the adaptor protein, MyD88, which recruits the serine/threoninekinases IRAK (IRAK-1 and IRAK-4). IRAKs form a complex with TRAF6, whichthen interacts with various molecules that participate in transducingthe TLR signal. These molecules and other TLRS signal transductionpathway components stimulate the activity of transcription factors, suchas fos, jun and NF-kB, and the corresponding induction of gene productsof fos-, jun- and NF-kB-regulated genes, such as, for example, IL-6,TNF-alpha, CXCL1, CXCL2 and CCL20. Typically, the flagellin polypeptideof the present invention comprises the domains of flagellin involved inTLRS signaling. The term “domain of flagellin” includes naturallyoccurring domain of flagellin and function conservative variantsthereof. “Function conservative variants” are those in which a givenamino acid residue in a protein or enzyme has been changed withoutaltering the overall conformation and function of the polypeptide,including, but not limited to, replacement of an amino acid with onehaving similar properties (such as, for example, polarity, hydrogenbonding potential, acidic, basic, hydrophobic, aromatic, and the like).Amino acids other than those indicated as conserved may differ in aprotein so that the percent protein or amino acid sequence identitybetween any two proteins of similar function may vary and may be, forexample, from 70% to 99%. Thus a “function-conservative variant” alsoincludes a polypeptide which has at least 70% amino acid identity withthe native sequence of flagellin or fragment thereof. According to theinvention a first amino acid sequence having at least 70% of identitywith a second amino acid sequence means that the first sequence has 70;71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88;89; 90; 91; 92; 93; 94; 95; 96; 97; 98; or 99, or 100% of identity withthe second amino acid sequence. In the same manner a first amino acidsequence having at least 90% of identity with a second amino acidsequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96;97; 98; or 99, or 100% of identity with the second amino acid sequence.Amino acid sequence identity is preferably determined using a suitablesequence alignment algorithm and default parameters, such as BLAST P(Karlin and Altschul, 1990). The domains of flagellin that are involvedin TLRS signaling are well known in the art, see for example Smith etal. (2003) Nat. Immunol. 4: 1247-1253 (e.g., amino acids 78-129, 135-173and 394-444 of S. typhimurium flagellin or homologs or modified formsthereof).

Examples of flagellin polypeptides include but are not limited to thosedescribed in U.S. Pat. Nos. 6,585,980; 6,130,082; 5,888,810; 5,618,533;and 4,886,748; U.S. Patent Publication No. US 2003/0044429 A1; and inthe International Patent Application Publications n° WO 2008097016 andWO 2009156405 which are incorporated by reference. An exemplary E. coliO157:H7 flagellin is SEQD ID NO:1. An exemplary S. typhimurium flagellinis SEQ ID NO:2 or SEQ ID NO:3.

In some embodiments, amino acid sequences having at least 70% ofidentity with SEQ ID NO: 1 SEQ ID NO:2 or SEQ ID NO:3 can be used asflagellin polypeptides according to the invention. In some embodiments,amino acid sequences having at least 90% of identity with SEQ ID NO: 1SEQ ID NO:2 or SEQ ID NO:3 can be used as flagellin polypeptidesaccording to the invention. In some embodiments, amino acid sequenceshaving at least 70% of identity with SEQ ID NO:3 can be used asflagellin polypeptides according to the invention provided that theresidues 89-96 (i.e. the residues that are involved in TLRS detection)are not mutated (i.e. not substituted or not deleted). In someembodiments, amino acid sequences having at least 90% of identity withSEQ ID NO: 1 SEQ ID NO:2 or SEQ ID NO:3 can be used as flagellinpolypeptides according to the invention provided that the residues 89-96(i.e. the residues that are involved in TLRS detection) are not mutated(i.e. not substituted or not deleted).

In some embodiments, the present encompasses the use of the flagellinrecombinant polypeptides described in the International PatentApplication n° WO 2009156405 which is incorporated by reference in itsentirely.

In some embodiments, the flagellin polypeptide of the present inventioncomprises: a) a N-terminal peptide having at least 90% amino acididentity with the amino acid sequence starting from the amino acidresidue located at position 1 of SEQ ID NO:3 and ending at an amino acidresidue selected from the group consisting of any one of the amino acidresidues located at positions 99 to 173 of SEQ ID NO:3 ; and b) aC-terminal peptide having at least 90% amino acid identity with theamino acid sequence starting at an amino acid residue selected from thegroup consisting of any one of the amino acid residues located atpositions 401 to 406 of SEQ ID NO:3 and ending at the amino acid residuelocated at position 494 of SEQ ID NO:3, wherein: the said N-terminalpeptide is directly linked to the said C-terminal peptide, or the saidN-terminal peptide and the said C-terminal peptide are indirectlylinked, one to the other, through a spacer chain. In some embodiments,said N-terminal peptide is selected from the group consisting of theamino acid sequences 1-99, 1-137, 1-160 and 1-173 of SEQ ID NO:3. Insome embodiments, said C-terminal peptide is selected from the groupconsisting of the amino acid sequences 401-494 and 406-494 of SEQ ID NO:3. In some embodiments, said N-terminal and C-terminal peptides consistof the amino acid sequences 1-173 and 401-494 of SEQ ID NO: 3,respectively. In some embodiments, said N-terminal and C-terminalpeptides consist of the amino acid sequences 1-160 and 406-494 of SEQ IDNO: 3, respectively. In some embodiments, said N-terminal and C-terminalpeptides consist of the amino acid sequences 1-137 and 406-494 of SEQ IDNO: 3, respectively. In some embodiments, said N-terminal peptide andthe said C-terminal peptide are indirectly linked, one to the other,through an intermediate spacer chain consisting of aNH2-Gly-Ala-Ala-Gly-COOH (SEQ ID NO: 4) peptide sequence. In someembodiments, the asparagine amino acid residue located at position 488of SEQ ID NO: 3 is replaced by a serine. In some embodiments, theflagellin polypeptide as above described comprises an additionalmethionine residue at the N-terminal end.

The flagellin polypeptide of the present invention is produced by anymethod well known in the art. In some embodiments, the flagellinpolypeptide of the present invention is typically recombinantly producedby recombinant cells that have been transfected with a nucleic acid thatencodes its amino acid sequence and allows its effective productionwithin the transfected cells. The nucleic acid sequence encoding theflagellin polypeptide of the invention, may be inserted into areplicable vector for cloning (amplification of the DNA) or forexpression. Various vectors are publicly available. The vector may, forexample, be in the form of a plasmid, cosmid, viral particle, or phage.The appropriate nucleic acid sequence may be inserted into the vector bya variety of procedures. In general, DNA is inserted into an appropriaterestriction endonuclease site(s) using techniques known in the art.Vector components generally include, but are not limited to, one or moreof a signal sequence if the sequence is to be secreted, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence. Construction of suitablevectors containing one or more of these components employs standardligation techniques that are known to the skilled artisan. Expressionand cloning vectors will typically contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up the nucleicacid encoding the flagellin polypeptide of the invention such as DHFR orthymidine kinase. An appropriate host cell when wild-type DHFR isemployed is the CHO cell line deficient in DHFR activity. Expression andcloning vectors usually contain a promoter operably linked to thenucleic acid sequence encoding the flagellin polypeptide to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known. Promoters suitable for use with prokaryotic hosts includethe beta-lactamase and lactose promoter systems, alkaline phosphatase, atryptophan (trp) promoter system, and hybrid promoters such as the tacpromoter. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S. D.) sequence operably linked to the DNA encoding theflagellin polypeptide of the invention. Host cells are transfected ortransformed with expression or cloning vectors described herein forflagellin polypeptide production and cultured in conventional nutrientmedia modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.The culture conditions, such as media, temperature, pH, and the like,can be selected by the skilled artisan without undue experimentation. Ingeneral, principles, protocols, and practical techniques for maximizingthe productivity of cell cultures can be found in Mammalian CellBiotechnology: A Practical Approach, M. Butler, ed. (IRL Press, 1991).Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include, but are not limited to, eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31, 446); E. coliX1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325); and K5772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain SIN41 of Salmonella typhimurium (fliC fljB), isparticularly interesting for the production of flagellin polypeptides ofthe invention, since these prokaryotic host cells do not secrete anyflagellins (Proc Natl Acad Sci USA. 2001; 98:13722-7). Howeverflagellins are secreted through specialized secretion system: the socalled “Type III secretion system”. Interestingly, strain SIN41 producesall components of the type III secretion system required for optimalflagellin secretion. Cloning sequence coding new flagellin peptidesunder fliC promoter enables secretion in large amounts of the flagellinpolypeptides of interest in strain SIN41. Strain W3110 is alsointeresting because it is a common host strain for recombinant DNAproduct fermentations. Preferably, the host cell secretes minimalamounts of proteolytic enzymes. For example, strain W3110 may bemodified to effect a genetic mutation in the genes encoding proteinsendogenous to the host, with examples of such hosts including E. coliW3110 strain 1A2, which has the complete genotype tonA; E. coli W3110strain 9E4, which has the complete genotype tonA ptr3; E. coli W31 10strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3phoA E15 (argF-lac)169 degP ompT kan.sup.r; E. coli W31 10 strain 37D6,which has the complete genotype tona ptr3 phoA E15 (argF-lac)169 degPompT rbs7 ilvG kan.sup.r; E. coli W31 10 strain 40B4, which is strain37D6 with a non-kanamycin resistant degP deletion mutation; and an E.coli strain having mutant periplasmic protease disclosed in U.S. Pat.No. 4,946,783 issued 7 Aug. 1990. The E. coli strains MG1655, MG1655AfimA-H or MKS12, a fliD- and -f/m>A-/-/-deleted MG1655 strain are alsointeresting candidates for production of recombinant flagellins assecreted proteins (Nat Biotechnol. 2005; (4):475-81). Alternatively, invitro methods of cloning, e.g., PCR or other nucleic acid polymerasereactions, are suitable. Flagellin polypeptide of the invention may berecovered from culture medium or from host cell lysates. Ifmembrane-bound, it can be released from the membrane using a suitabledetergent solution (e.g., TRITON-XTM. 100) or by enzymatic cleavage. Insome embodiments, the flagellin polypeptide is purified from thesupernatant of recombinant S. Typhimurium SIN41 (fliC fljB), asdisclosed in Nempont et al. (Nempont, C. C., D.; Rumbo, M.; Bompard, C.;Villeret, V.; Sirard, J. C. 2008. Deletion of flagellin's hypervariableregion abrogates antibody-mediated neutralization and systemicactivation of TLRS-dependent immunity. J Immunol 181:2036-2043.). Inparticular, Salmonella were grown in Luria-Bertani (LB) broth for 6-18hours at 37° C. with agitation. The supernatant was filtered andsaturated with 60% ammonium sulfate (Sigma Aldrich, USA). Theprecipitated materials were recovered by centrifugation, solubilizationin 20 mM Tris/HCl pH7.5 and then dialysis. The proteins were furtherpurified by successive rounds of hydroxyapatite, anion exchange, andsize exlusion chromatography (Bio-Rad Laboratories, USA; GE Healthcare,Sweden). Lastly, the proteins were depleted of lipopolysaccharide (LPS)using a polymyxin B column (Pierce, USA). Using the Limulus assay(Associates of Cape Cod Inc., USA), the residual LPS concentration wasdetermined to be less than 30 pg LPS per μg recombinant flagellin.Constructs encoding the flagellins may be generated by PCR and clonedinto the expression vector pET22b+. The plasmids can be introduced inEscherichia coli BL21 (DE3) and protein production can be induced byadding IPTG 1mM. After disruption on French press, the soluble fractionwas depleted of lipopolysaccharide (LPS) using Triton X-114 extraction.If flagellins are found in the insoluble fraction after theFrench-press, inclusion bodies are denatured in presence of Urea 8Mfollowed by dialysis and Triton X-114 extraction. The proteins can thenbe purified on anion exchange chromatography and gel filtration.Finally, proteins can be again depleted of LPS using a polymyxin Bcolumn (Pierce, USA).

In some embodiments, the antibiotic is selected from the groupconsisting of aminoglycosides, beta lactams, quinolones orfluoroquinolones, macrolides, sulfonamides, sulfamethaxozoles,tetracyclines, streptogramins, oxazolidinones (such as linezolid),rifamycins, glycopeptides, polymixins, lipo-peptide antibiotics.

Tetracyclines belong to a class that shares a four-membered ringstructure composed of four fused 6-membered (hexacyclic) rings. Thetetracyclines exhibit their activity by inhibiting the binding of theaminoacyl tRNA to the 30S ribosomal subunit in susceptible bacteria.Tetracyclines for use in the invention include chlortetracycline,demeclocycline, doxycycline, minocycline, oxytetracycline,chlortetracycline, methacycline, mecocycline, tigecycline, limecycline,and tetracycline. The tetracyclines are effective against many knownorganisms including a-hemolytic streptococci, nonhemolytic streptococci,gram negative bacilli, rickettsiae, spirochetes, Mycoplasma, andChlamydia.

Aminoglycosides are compounds derived from species of Streptomyces orMicomonospora bacteria and are primarily used to treat infections causedby gram-negative bacteria. Drugs belonging to this class all possess thesame basic chemical structure, i.e., a central hexose or diaminohexosemolecule to which two or more amino sugars are attached by a glycosidicbond. The aminoglycosides are bactericidal antibiotics that bind to the30S ribosome and inhibit bacterial protein synthesis. They are activeprimarily against aerobic gram-negative bacilli and staphylococci.Aminoglycoside antibiotics for use in the invention include amikacin(Amikin®), gentamicin (Garamycin®), kanamycin (Kantrex®), neomycin(Mycifradin®), netilmicin (Netromycin®), paromomycin (Humatin®),streptomycin, and tobramycin (TOBI Solution®, TobraDex®).

Macrolides are a group of polyketide antibiotic drugs whose activitystems from the presence of a macrolide ring (a large 14-, 15-, or16-membered lactone ring) to which one or more deoxy sugars, usuallycladinose and desosamine, are attached. Macrolides are primarilybacteriostatic and bind to the 50S subunit of the ribosome, therebyinhibiting bacterial synthesis. Macrolides are active against aerobicand anaerobic gram positive cocci (with the exception of enterococci)and against gram-negative anaerobes. Macrolides for use in the inventioninclude azithromycin (Zithromax®), clarithromycin (Biaxin®),dirithromycin (Dynabac®), erythromycin, clindamycin, josamycin,roxithromycin and lincomycin.

Ketolides belong to a class of semi-synthetic 14-membered ringmacrolides in which the erythromycin macrolactone ring structure and theD-desosamine sugar attached at position 5 are retained, however,replacing the L-cladinose5 moiety and hydroxyl group at position 3 isa3-keto functional group. The ketolides bind to the 23S rRNA, and theirmechanism of action is similar to that of macrolides (Zhanel, G. G., etal., Drugs, 2001; 61(4):443-98). The ketolides exhibit good activityagainst gram-positive aerobes and some gram-negative aerobes, andpossess excellent activity against Streptococcus spp. including mefA andermB-producing Streptococcus pneumoniae, and Haemophilus influenzae.Representative ketolides for use in the invention include telithromycin(formerly known as HMR-3647), HMR 3004, HMR 3647, cethromycin, EDP-420,and ABT-773.

Structurally, the quinolones possess a 1,4 dihydro-4-oxo-quinolinylmoiety bearing an essential carboxyl group at position 3. Functionally,the quinolones inhibit prokaryotic type II topoisomerases, namely DNAgyrase and, in a few cases, topoisomerase IV, through direct binding tothe bacterial chromosome. Quinolones for use in the invention spanfirst, second, third and fourth generation quinolones, includingfluoroquinolones. Such compounds include nalidixic acid, cinoxacin,oxolinic acid, flumequine, pipemidic acid, rosoxacin, norfloxacin,lomefloxacin, ofloxacin, enrofloxacin, ciprofloxacin, enoxacin,amifloxacin, fleroxacin, gatifloxacin, gemifloxacin, clinafloxacin,sitafloxacin, pefloxacin, rufloxacin, sparfloxacin, temafloxacin,tosufloxacin, grepafloxacin, levofloxacin, moxifloxacin, andtrovafloxacin. Additional quinolones suitable for use in the inventioninclude those described in Hooper, D., and Rubinstein, E., “QuinoloneAntimicrobial Agents, Vd Edition”, American Society of MicrobiologyPress, Washington D.C. (2004).

Drugs belonging to the sulfonamide class all possess a sulfonamidemoiety, SO2NH2, or a substituted sulfonamide moiety, where one 15 of thehydrogens on the nitrogen is replaced by an organic substituent.Illustrative N-substituents include substituted or unsubstitutedthiazole, pyrimidine, isoxazole, and other functional groups.Sulfonamide antibiotics all share a common structural feature, i.e.,they are all benzene sulfonamides, 20 meaning that the sulfonamidefunctionality is directly attached to a benzene ring. The structure ofsulfonamide antibiotics is similar to p-aminobenzoic acid (PABA), acompound that is needed in bacteria as a substrate for the enzyme,dihydropteroate synthetase, for the synthesis of tetrahydro-25 folicacid. The sulfonamides function as antibiotics by interfering with themetabolic processes in bacteria that require PABA, thereby inhibitingbacterial growth and activity. Sulfonamide antibiotics for use in theinvention include the following: mafenide, phtalylsulfathiazole,succinylsulfathiazole, sulfacetamide, sulfadiazine, sulfadoxine,sulfamazone, sulfamethazine, sulfamethoxazole, sulfametopirazine,sulfametoxypiridazine, sulfametrol, sulfamonomethoxine, sulfamylon,sulfanilamide, sulfaquinoxaline, sulfasalazine, sulfathiazole,sulfisoxazole, sulfisoxazole diolamine, and sulfaguanidine.

All members of beta-lactams possess a beta-lactam ring and a carboxylgroup, resulting in 55 similarities in both their pharmacokinetics andmechanism of action. The majority of clinically useful beta-lactamsbelong to either the penicillin group or the cephalosporin group,including cefamycins and oxacephems. The beta-lactams also include thecarbapenems and monobactams. Generally speaking, beta-lactams inhibitbacterial cell wall synthesis. More specifically, these antibioticscause ‘nicks’ in the peptidoglycan net of the cell wall that allow thebacterial protoplasm to flow from its protective net into thesurrounding hypotonic medium. Fluid then accumulates in the naked 65protoplast (a cell devoid of its wall), and it eventually bursts,leading to death of the organism. Mechanistically, beta-lactarns act byinhibiting D-alanyl-D-alanine transpeptidase activity by forming stableesters with the carboxyl of the open lactam ring attached to thehydroxyl group of the enzyme target site. Beta-lactams are extremelyeffective and typically are of low toxicity. As a group, these drugs areactive against many gram-positive, gram-negative and anaerobicorganisms. Drugs falling into this category include 2-(3-alanyl)clavam,2-hydroxymethylclavam, 7-methoxycephalosporin, epi-thienamycin,acetyl-thienamycin, amoxicillin, apalcillin, aspoxicillin, azidocillin,azlocillin, aztreonam, bacampicillin, blapenem, carbenicillin,carfecillin, carindacillin, carpetimycin A and B, cefacetril, cefaclor,cefadroxil, cefalexin, cefaloglycin, cefaloridine, cefalotin,cefamandole, cefapirin, cefatrizine, cefazedone, cefazolin,cefbuperazone, cefcapene, cefdinir, cefditoren, cefepime, cefetamet,cefixime, cefinenoxime, cefinetazole, cefminox, cefmolexin, cefodizime,cefonicid, cefoperazone, ceforamide, cefoselis, cefotaxime, cefotetan,cefotiam, cefoxitin, cefozopran, cefpiramide, cefpirome, cefpodoxime,cefprozil, cefquinome, cefradine, cefroxadine, cefsulodin, ceftazidime,cefteram, ceftezole, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime,cephalosporin C, cephamycin A, cephamycin C, cephalothin, chitinovorinA, chitinovorin B, chitinovorin C, ciclacillin, clometocillin,cloxacillin, cycloserine, deoxy pluracidomycin B and C, dicloxacillin,dihydro pluracidomycin C, epicillin, epithienamycin D, E, and F,ertapenem, faropenem, flomoxef, flucloxacillin, hetacillin, imipenem,lenampicillin, loracarbef, mecillinam, meropenem, metampicillin,meticillin (also referred to as methicillin), mezlocillin, moxalactam,nafcillin, northienamycin, oxacillin, panipenem, penamecillin,penicillin G, N, and V, phenethicillin, piperacillin, povampicillin,pivcefalexin, povmecillinam, pivmecillinam, pluracidomycin B, C, and D,propicillin, sarmoxicillin, sulbactam, sultamicillin, talampicillin,temocillin, terconazole, thienamycin, andticarcillin.

Over 400 natural antimicrobial peptides have been isolated andcharacterized. Based on chemical structure, these peptides may beclassified into two main groups: linear and cyclic (R.E. Hancock et al,Adv. Microb. Physiol., 1995, 37: 135-137; H. Kleinkauf et al., Criti.Rev. Biotechnol., 198, 8: 1-32; D. Perlman and M. Bodansky, Annu. Rev.Biochem., 1971, 40: 449-464). The mode of action for the majority ofthese peptides (both linear and cyclic) is believed to involve membranedisruption, leading to cell leakage (A. Mor, Drug Develop. Res., 2000,50: 440-447). The linear peptides, such as magainins and melitting,exist mainly as a-helical amphipathic structures (containing segregatedhydrophobic and hydrophilic moieties), or as β-helices as found ingramicidin A (GA). Cyclic peptides, which mainly adopt an amphipaticβ-sheet structures can be further divided into two subgroups: thosecontaining disulfide bonds, such as tachyplesin, and those that do not,such as gramicidin S (D. Audreu and L. Rivas, Biopolymers, 1998, 47:415-433). Peptide antibiotics also fall into two classes:non-ribosomally synthesized peptides, such as the gramicicins,polymyxins, bacitracins, glycopeptides, etc., and ribosomallysynthesized (natural) peptides. The former are often drasticallymodified and are largely produced by bacteria, whereas the latter areproduced by all species of life (including bacteria) as a majorcomponent of the natural host defense molecules of these species. Incertain embodiments, the peptide antibiotic is a lipopeptide antibioticsuch as colistin, daptomycin, surfactin, friulimicin, aculeacin A,iturin A, and tsushimycin. Colistin (also called Colimycin) is apolymixin antibiotic discovered more than 50 years ago. It is a cycliclipopeptide antibiotic which penetrates the cell wall of Gram negativebacteria by self-induced mechanism by chelating divalent ions. Colistindestabilizes the wall and can insinuate into it. Colistin basicallyperforates the cell wall, causing distortion of this structure and therelease of intracellular constituents. Increasing multidrug resistancein Gram-negative bacteria, in particular Pseudomonas aeruginosa,Acinetobacter baumannii, and Klebsiella pneumoniae, presents a criticalproblem. Limited therapeutic options have forced infectious diseaseclinicians and microbiologists to reappraise the clinical application ofColistin. Colistin is associated with neurotoxicity and nephrotoxicity.Dosage regimen and novel formulation may be an answer to address thetoxicity issue.

In some embodiments, the flagellin polypeptide is used in combinationwith amoxicillin.

In some embodiments, the flagellin polypeptide is used in combinationwith bactrim® which contains both sulfamethoxazole and trimethoprime.

In some embodiments, the flagellin polypeptide and the antibiotic are tobe used simultaneous or sequentially within a given time. The antibioticcan be applied in either order, e.g. the antibiotic can be applied firstand then the flagellin polypeptide can be applied or vice versa. It isobvious that when a composition comprising both the antibiotic andflagellin polypeptide is used both components will be applied at thesame time by the same routes or by different routes of administration.For example, the antibiotic may be administered to the subject via theoral route and the flagellin polypeptide is administered to the subjectvia the intravenous route or via the intranasal route.

By a “therapeutically effective amount” is meant a sufficient amount ofthe flagellin polypeptide and/or antibiotic for the treatment of abacterial superinfection post influenza at a reasonable benefit/riskratio applicable to any medical treatment. It will be understood thatthe total daily usage of the compounds and compositions of the presentinvention will be decided by the attending physician within the scope ofsound medical judgment. The specific therapeutically effective doselevel for any particular subject will depend upon a variety of factorsincluding the age, body weight, general health, sex and diet of thesubject; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificpolypeptide employed; and like factors well known in the medical arts.For example, it is well known within the skill of the art to start dosesof the compound at levels lower than those required to achieve thedesired therapeutic effect and to gradually increase the dosage untilthe desired effect is achieved. However, the daily dosage of theproducts may be varied over a wide range from 0.01 to 1,000 mg per adultper day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0,2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the activeingredient for the symptomatic adjustment of the dosage to the subjectto be treated. A medicament typically contains from about 0.01 mg toabout 500 mg of the active ingredient, preferably from 1 mg to about 100mg of the active ingredient. An effective amount of the drug isordinarily supplied at a dosage level from 0.0002 mg/kg to about 20mg/kg of body weight per day, especially from about 0.001 mg/kg to 7mg/kg of body weight per day.

Typically the active ingredient of the present invention (i.e. theflagellin polypeptide and/or antibiotic) is combined withpharmaceutically acceptable excipients, and optionally sustained-releasematrices, such as biodegradable polymers, to form pharmaceuticalcompositions. The term “Pharmaceutically” or “pharmaceuticallyacceptable” refers to molecular entities and compositions that do notproduce an adverse, allergic or other untoward reaction whenadministered to a mammal, especially a human, as appropriate. Apharmaceutically acceptable carrier or excipient refers to a non-toxicsolid, semi-solid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The carrier can also be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), suitable mixtures thereof, and vegetables oils. The properfluidity can be maintained, for example, by the use of a coating, suchas lecithin, by the maintenance of the required particle size in thecase of dispersion and by the use of surfactants. The prevention of theaction of microorganisms can be brought about by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminium monostearate and gelatin. In thepharmaceutical compositions of the present invention, the activeingredients of the invention can be administered in a unitadministration form, as a mixture with conventional pharmaceuticalsupports. Suitable unit administration forms comprise oral-route formssuch as tablets, gel capsules, powders, granules and oral suspensions orsolutions, sublingual and buccal administration forms, aerosols,implants, subcutaneous, transdermal, topical, intraperitoneal,intramuscular, intravenous, subdermal, transdermal, intrathecal andintranasal administration forms and rectal administration forms. In someembodiments, the pharmaceutical composition of the invention isadministered topically (i.e. in the respiratory tract of the subject).Therefore, the compositions can be formulated in the form of a spray,aerosol, solution, emulsion, or other form well-known to one of skill inthe art. If the method of the invention comprises intranasaladministration of a composition, the composition can be formulated in anaerosol form, spray, mist or in the form of drops. In particular, theactive ingredients for use according to the present invention can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebuliser, with the use of a suitable propellant(e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridges(composed of, e.g., gelatin) for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

FIGURES

FIG. 1: Flagelin stimulates proinflammatory gene expression duringInfluenza A virus infection. C57BL/6 mice were infected intranasallywith Influenza A virus (30 PFU). Seven or 14 days after viral infection,mice were treated intranasally with 2.5 μg flagellin FliC_(Δ174-400).Two hours post-flagellin stimulation, lungs were collected for analysisof transcript levels by quantitative PCR. Results are given asmeans±standard errors of the mean (n=4). (A-C) Messenger RNA levels areexpressed relative to those of the mock group (uninfected anduntreated), arbitrary set to 1. (D) Virus RNA are expressed compared toa housekeeping gene expression (Bactin) and the limit of detection wasset arbitrary to 1 with an uninfected C57BL/6 mice. Statisticalsignificance was determined with the non parametric test of Mann-Whitney(*: p<0.05).

FIG. 2: The AMX/FliC_(Δ174-400) treatment protect Influenza A virus-S.pneumoniae co-infected mice. (A) C57BL/6 mice were infected withinfluenza A virus H3N2 (30 PFU). Seven days later, mice were infectedwith S. pneumoniae (10³ CFU). Twelve hours and 42 h later, animals weretreated intragastrically with 5 μg AMX and intranasally with 2.5 μgflagellin FliC_(Δ174-400). At 60 h, bacterial counts in lung (B) andspleen (C) were determined by measuring CFU per tissue. Each dotrepresents CFU for an individual mouse. The solid line represents thethreshold of detection. Statistical significance was determined with thenon parametric test of Mann-Whitney (*: p<0.05 and **: p<0.01).

EXAMPLE

Materials & Methods

Bacterial Preparation

Streptococcus pneumoniae serotype 1 (clinical isolate E1586) wasobtained from the National Reference Laboratory—Ministry of Health,Uruguay. Working stocks were prepared as follows: Todd Hewitt YeastBroth (THYB) (Sigma-Aldrich—Saint-Louis, Mo.) was inoculated with freshcolonies grown in blood-agar plates, and incubated at 37° C. untilOD600nm of 0.7-0.9 units. Cultures were stored at −80° C. inTHYB+glycerol 12% (vol/vol) up to 3 months. For mouse infection, workingstocks were thawed and washed with sterile Dulbecco's Phosphate-BufferedSaline (DPBS; Gibco—Grand Island, N.Y.) and diluted to the appropriateconcentration. Number of bacteria in stocks was confirmed by platingserial dilutions onto blood agar plates.

Mouse Model of Infection

Female BALB/c (6-8 weeks old) mice were obtained from Janvierlaboratories (St. Berthevin, France). Animals were maintained inindividually ventilated cages and handled in a vertical laminar flowcabinet (class II A2, ESCO—Hatboro, Pa.). All experiments complied withcurrent national and institutional regulations and ethical guidelines(B59-350009—Institut Pasteur de Lille). Mice were anaesthetized byintraperitoneal (i.p.) injection of 1.25 mg ketamine (Imalgène,Merial—Lyon, France) plus 0.25 mg xylazine (Rompun, BayerHealthCare—Loos, France) in 250 μl DPBS. Mice were infected i.n. with 50μl of D-PBS containing 30 PFU of the highly-pathogenic murine adaptedH3N2 Influenza A virus strain Scotland/20/74. Seven or 14 dayspost-viral challenge, mice were infected i.n. with 30 μl of D-PBScontaining 10³ CFU of S. pneumoniae.

Flagellin and Antibiotic Administration

The constructs encoding the recombinant flagellins FliC_(Δ174-400)(harboring a carboxy-terminal histidine Tag) and rFliC (harboring anamino-terminal histidine Tag) were generated by PCR and cloned into theexpression vector pET22b+. The recombinant flagellins were produced asfollows. The plasmids were introduced in Escherichia coli BL21(DE3) andprotein production was induced by adding IPTG 1 mM. After disruption onFrench press, the soluble fraction was depleted of lipopolysaccharide(LPS) using Triton X-114 extraction as described previously. Theproteins were purified successively on nickel affinity chromatography,anion exchange chromatography and gel filtration by Fast protein liquidchromatography (GE Healthcare). Finally, proteins were again depleted ofLPS using a polymyxin B column (Pierce, USA). Using the Limulus assay(Associates of Cape Cod Inc., USA), the residual LPS concentration wasdetermined to be less than 20 pg LPS per μg recombinant flagellin.Flagellins were heated for 10 min at 65° C. before use to ensure thatproteins are mostly monomers. Flagellins in 30 μl DPBS wereadministrated i.n. under light anesthesia by inhalation of isoflurane(Axience—Pantin, France) using an anaesthesia non-rebreathing system(DRE-Compact 150, DRE Veterinary—Louisville, Ky.). Infected mice weretreated by a suboptimal dose of amoxicillin (AMX) (5 μg in 200 μl water;amoxicillin VERTANAL™, Sigma-Aldrich—Saint-Louis, Mo.) intragastrically(i.g.) using a plastic tube feed (V0104030, ECIMED—Boissy-St-Léger,France). This represents a dose of AMX of 250 μg/kg for 6 to 8 week oldmice.

Determination of Bacterial Load in Lungs and Spleen

Mice were sacrificed at different time points after infection by i.p.injection of 5.47 mg of sodium pentobarbital (CEVA Santé animale,Libourne, France) in 100 μl DPBS. Lungs and spleen were collected atselected time points after infection and homogenized with an UltraTurraxhomogenizer (IKA-Werke, Staufen, Germany). Viable counts were determinedby plating serial dilutions onto blood-agar plates.

Gene Expression Quantification by Real-Time RT-PCR

Total lung RNAs were extracted with the Nucleospin RNA II kit (MachereyNagel—Hoerdt, France) and reverse-transcribed with the High-CapacitycDNA Archive Kit (Applied Biosystems—Foster city, Canada). The resultingcDNA was amplified using SYBR Green-based real-time PCR on a 7300 RealTime PCR System (Applied Biosystems). Primers specific for referencegene ActB, and chemokine-encoding genes Ccl20 and Cxcl1 were describedpreviously [20]. Relative mRNA levels (2^(−ΔΔCt)) were determined bycomparing first, the PCR cycle thresholds (Ct) for the gene of interestand Δctb (ΔCt) and second, the ΔCt values for treated and referencegroup (ΔΔCt), as described previously [20].

Statistical Analysis

Results are expressed as median±range. Statistical differences wereanalyzed using the Mann-Whitney test (GraphPad Prism 5.0) and wereconsidered to be significant for p values<0.05.

Results:

We investigated the effectiveness of a combination therapy consisting offlagellin+antibiotic on IAV-infected animals. First, we defined whetherflagellin is able to promote signaling in IAV-infected animals. We foundthat intranasal treatment with FliC_(Δ174-400) was able to furtherincrease the transcription of immune mediators in the context ofIAV-infection both in the acute and resolution phases, e.g. 7 and 14days post-infection (FIG. 1). We selected to assess the combinationtherapy in animals in acute infection. For this purpose, animalsinfected with IAV for 7 days were infected with S. pneumoniae andtreated with AMX and flagellin (FIG. 2). Our data showed that thecombination therapy was highly effective to increase the therapeuticindex of AMX both in lungs and spleen.

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

1. A method of treating a bacterial superinfection post-influenza in asubject in need thereof comprising administering to the subject atherapeutically effective amount of a flagellin polypeptide.
 2. Themethod of claim 1 wherein the bacterial superinfection is mediated by atleast one organism selected from the group consisting of Streptococcuspneumoniae; Staphylococcus aureus; Haemophilus influenza, Myoplasmaspecies and Moraxella catarrhalis.
 3. The method of claim 1 wherein thesubject is selected from the group consisting of subjects who are atleast 50 years old, subjects who reside in chronic care facilities,subjects who have chronic disorders of the pulmonary or cardiovascularsystem, subjects who required regular medical follow-up orhospitalization during the preceding year because of chronic metabolicdiseases, renal dysfunction, hemoglobinopathies, or immunosuppression,children less than 14 years of age, patients between 6 months and 18years of age who are receiving long-term aspirin therapy, and women whowill be in the second or third trimester of pregnancy during theinfluenza season.
 4. The method of claim 1 wherein the flagellinpolypeptide has at least 70% of identity with SEQ ID NO:1, SEQ ID NO:2or SEQ ID NO:3.
 5. The method of claim 1 wherein the flagellinpolypeptide comprises: a) a N-terminal peptide having at least 90% aminoacid identity with the amino acid sequence starting from the amino acidresidue located at position 1 of SEQ ID NO:3 and ending at an amino acidresidue selected from the group consisting of any one of the amino acidresidues located at positions 99 to 173 of SEQ ID NO:3; and b) aC-terminal peptide having at least 90% amino acid identity with theamino acid sequence starting at an amino acid residue selected from thegroup consisting of any one of the amino acid residues located atpositions 401 to 406 of SEQ ID NO:3 and ending at the amino acid residuelocated at position 494 of SEQ ID NO:3, wherein: the said N-terminalpeptide is directly linked to the said C-terminal peptide, or the saidN-terminal peptide and the said C-terminal peptide are indirectlylinked, one to the other, through a spacer chain.
 6. The method of claim5 wherein said N-terminal peptide is selected from the group consistingof the amino acid sequences 1-99, 1-137, 1-160 and 1-173 of SEQ ID NO:3.7. The method of claim 5 wherein said C-terminal peptide is selectedfrom the group consisting of the amino acid sequences 401-494 and406-494 of SEQ ID NO:3.
 8. The method of claim 5 wherein said N-terminaland C-terminal peptides consist of the amino acid sequences 1-173 and401-494 of SEQ ID NO:3, respectively.
 9. The method of claim 5 whereinsaid N-terminal and C-terminal peptides consist of the amino acidsequences 1-160 and 406-494 of SEQ ID NO:3, respectively.
 10. The methodof claim 5 wherein said N-terminal and C-terminal peptides consist ofthe amino acid sequences 1-137 and 406-494 of SEQ ID NO:3, respectively.11. The method of claim 5 wherein said N-terminal peptide and saidC-terminal peptide are indirectly linked, one to the other, through anintermediate spacer chain consisting of a NH2-Gly-Ala-Ala-Gly-COOH (SEQID NO:4) peptide sequence.
 12. The method of claim 5 wherein theasparagine amino acid residue located at position 488 of SEQ ID NO:3 isreplaced by a serine.
 13. The method of claim 16, wherein the antibioticis selected from the group consisting of aminoglycosides, beta lactams,quinolones, fluoroquinolones, macrolides, sulfonamides,sulfamethaxozoles, tetracyclines, streptogramins, and oxazolidinones,rifamycins, glycopeptides, polymixins, and Lipo-peptide antibiotics. 14.The method of claim 16, wherein the antibiotic is amoxicillin.
 15. Themethod of claim 16, wherein the antibiotic contains bothsulfamethoxazole and trimethoprime.
 16. The method of claim 1, whereinthe flagellin polypeptide is administered in combination with at leastone antibiotic